USF1 and USF2 are ubiquitous transcription factors of the basic helix-loop-helix leucine zipper family. They form homo- and heterodimers and recognize a CACGTG motif termed E box. In the liver, USF binding activity is mainly accounted for by the USF1/USF2 heterodimer, which binds in vitro the glucose/carbohydrate response elements (GlRE/ChoRE) of glucose-responsive genes. To assign a physiological role of USFs in vivo, we have undertaken the disruption of USF1 and USF2 genes in mice. We present here the generation of USF1-deficient mice. In the liver of these mice, we demonstrate that USF2 remaining dimers can compensate for glucose responsiveness, even though the level of total USF binding activity is reduced by half as compared with wild type mice. The residual USF1 binding activity was similarly reduced in the previously reported USF2 -/- mice in which an impaired glucose responsiveness was observed (Vallet, V. S., Henrion, A. A., Bucchini, D., Casado, M. , Raymondjean, M., Kahn, A., and Vaulont, S. (1997) J. Biol. Chem. 272, 21944-21949). Taken together, these results clearly suggest differential transactivating efficiencies of USF1 and USF2 in promoting the glucose response. Furthermore, they support the view that USF2 is the functional transactivator of the glucose-responsive complex.

In the liver, transcription of several genes encoding lipogenic and glycolytic enzymes, in particular the gene for fatty acid synthase (FAS), is known to be stimulated by dietary carbohydrates. The molecular dissection of the FAS promoter pointed out the critical role of an E box motif, located at position ؊65 with respect to the start site of transcription, in mediating the glucose-and insulin-dependent regulation of the gene. Upstream stimulatory factors (USF1 and USF2) and sterol response element binding protein 1 (SREBP1) were shown to be able to interact in vitro with this E box. However, to date, the relative contributions of USFs and SREBP1 ex vivo remain controversial. To gain insight into the specific roles of these factors in vivo, we have analyzed the glucose responsiveness of hepatic FAS gene expression in USF1 and USF2 knock-out mice. In both types of mouse lines, defective in either USF1 or USF2, induction of the FAS gene by refeeding a carbohydrate-rich diet was severely delayed, whereas expression of SREBP1 was almost normal and insulin response unchanged. Therefore, USF transactivators, and especially USF1/ USF2 heterodimers, seem to be essential to sustain the dietary induction of the FAS gene in the liver.Fatty acid synthase (FAS) 1 plays a central role in de novo lipogenesis in mammals, catalyzing all reaction steps in the conversion of acetyl-CoA and malonyl-CoA to palmitate. As for many other lipogenic and glycolytic genes involved in maintenance of energy balance, the expression of the FAS gene is highly dependent on nutritional conditions in liver and adipose tissue. Expression of the gene is barely detectable in starved animals and is stimulated by refeeding a high carbohydrate, fat-free diet (for review see Refs. 1 and 2). This fasting/refeeding transition is accompanied in vivo by an increased circulating insulin level, and it is often difficult to differentiate the effects of insulin from those of carbohydrate metabolism in mediating the regulation of gene expression. To date, the role of insulin, either direct or indirect, in mediating FAS-activated gene expression, is still disputed. It has been proposed that the effects of insulin could be only indirect (3-5), being permissive to allow for effective glucose metabolism (1). In contrast, others have provided evidence for a direct involvement of insulin both ex vivo (6) and in vivo (7). In this respect, Sul and co-workers (8) reported, by transfection experiments, that the region between Ϫ71/Ϫ50 was responsible for mediating the effects of insulin on the rat FAS promoter (see Fig. 1). They further demonstrated that upstream stimulatory factors (USFs), USF1 and USF2, were major components of the complex binding to this region (called IRS for insulin response sequence) (9). USFs are ubiquitous basic helix-loop-helix-leucine zipper (b-HLH-Zip) transcription factors able to interact as homo-and/or heterodimers on E boxes of CANNTG sequence (for review see Ref. 10). The FAS promoter IRS contains such an E box at position Ϫ65 (Fig. 1). Mut...

Upstream stimulatory factors (USF) 1 and 2 belong to the Myc family of transcription factors characterized by a basic/helix loop helix/leucine zipper domain responsible for dimerization and DNA binding. These ubiquitous factors form homo-and heterodimers and recognize in vitro a CACGTG core sequence termed E box. Through binding to E boxes of target genes, USF factors have been demonstrated to activate gene transcription and to enhance expression of some genes in response to various stimuli. In particular, in the liver USF1 and USF2 have been shown to bind in vitro glucose/carbohydrate response elements of glycolytic and lipogenic genes and have been proposed, from ex vivo experiments, to be involved in their transcriptional activation by glucose. However, the direct involvement of these factors in gene expression and nutrient gene regulation in vivo has not yet been demonstrated. Therefore, to gain insight into the specific role of USF1 and USF2 in vivo, and in particular to determine whether the USF products are required for the response of genes to glucose, we have created, by homologous recombination, USF2 ؊/؊ mice. In this paper, we provide the first evidence that USF2 proteins are required in vivo for a normal transcriptional response of L-type pyruvate kinase and Spot 14 genes to glucose in the liver.

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